Extraction and Characterization of a Three Known Component Mixture

To identify each recovered fraction purity and identify FTIR spectroscopy was collected, TLC was conducted, and MP for benzoic acid was concluded.

TLC was used to determine the purity of each component using an ideal eluent system that most efficiently separates every compound on a TLC plate. To determine the idea eluent five different mixtures of hexane and ethyl acetate were tested with pure samples of O-toluidine, Benzoic acid, and Anisole, and a mixture of each three pure compounds on the TLC plate to compare and conclude which eluent system separated the three components from the mixture most efficiently. This was determined by comparing the spots in each lane of a TLC plate for each eluent. Table 2: Ideal Eluent System shows how the ideal eluent system was chosen using only the Mixture to compare each eluent system.

Table 2: Ideal Eluent System

Eluent System

Compounds of Mixture

Distance compound traveled (cm-1)

Deviation

100% Ethyl Acetate

Anisole, Benzoic acid, O-toluidine

3.2, 2.7, 3.0

0.3

1:3 Ethyl Acetate/ Hexane

Anisole, Benzoic acid, O-toluidine

0.7, 1.5, 3.1

0.8

1:1 Ethyl Acetate/ Hexane

Anisole, Benzoic acid, O-toluidine

1.5, 2.0, 2.5

0.5

3:1 Ethyl Acetate/ Hexane

Anisole, Benzoic acid, O-toluidine

0, 0.4, 3.0

0.6

100% Hexane

Anisole, Benzoic acid, O-toluidine

1.5, 2.6, 3.2

0.6

After analysis, it is clear that the ideal eluent system is 1:1 ratio of Hexane and ethyl acetate as the deviation between the distance each mixture moved was exactly 0.5 every time which is the most constant deviation compared to the other eluent systems. This ideal eluent was then used to analyze the extraction samples by conducting the same experiment only with the extracted fraction compounds.

Retention factor (Rf) values of each TLC plate were then calculated. A compound with low polarity will have a larger Rf value than a polar compound when compared. Thus, Rf values can be used to identify a compound and its purity by comparing an unknown compound (extraction factions) to the known pure compounds. If the Rf values are the same, the two compounds are most likely the same. Rf values were determined by subtracting the distance (cm) traveled by the sample by the distance (cm) traveled by the eluent. Table 3: Rf values of Ideal Eluent TLC Plate shows the Rf values of the extracted fraction TLC Plates.

After analysis, it is clear that the ideal eluent system is 1:1 ratio of Hexane and ethyl acetate as the deviation between the distance each mixture moved was exactly 0.5 every time which is the most constant deviation compared to the other eluent systems. This ideal eluent was then used to analyze the extraction samples by conducting the same experiment only with the extracted fraction compounds.

Table 3: Rf values of Ideal Eluent TLC Plate

Eluent System

Compound

Distance Traveled (cm)

Rf Value

1:1 Ethyl Acetate/ Hexane

Eluent

Pure Anisole

Pure Benzoic acid

Pure O-toluidine

3.5

1.5

1.3

2.0

N/A

0.43

0.37

0.57

1:1 Ethyl Acetate/ Hexane

Eluent

Anisole Fraction

Benzoic acid Fraction

O-toluidine Fraction

3.2

1.5

1.7

1.7

N/A

0.47

0.53

0.53

Based off of Rf values it is clear that the 1:1 ratio of Ethyl Acetate/ Hexane efficiently separated every three compounds from the mixture as the Rf values of the pure samples compare relatively closely to the Rf values of the fractions.

FTIR spectrums of each isolated compound from the Acid-Base were then collected and analyzed, and the major non-fingerprint peaks in each fraction are listed in Table 4: FTIR Fingerprint Peaks to determine the purity of each extraction fraction.

Table 4: FTIR Fingerprint Peaks.

Compound

FTIR Band (cm-1)

Anisole Fraction

2931.97, 2862.49 sp3-sp3 C-H stretch

1634.00sp3-sp3 N-H

1498.20 sp2-sp2 C-C aromatic

1311.42 sp3-sp3 C-O stretch

Benzoic acid Fraction

3067.77, 2935.13, 2862.49 sp2-sp3 C-H stretch

1965.60, 1915.07 sp2-sp2 C=O stretch

1675.06, 1602.42, 1586.63 sp2-sp2 C=C stretch (in-ring)

O-toluidine Fraction

3462.53, 3358.31, 1618.21 sp3-sp3 N-H stretch

3020.40, 2928.81, 2862.49 sp3-sp3 C-H stretch

1583.47 sp2-sp2 C=C stretch

By comparing the FTIR band values of the extracted fractions to correlated structures, the identity of each fraction and its relative purity was concluded. Anisole Fraction was partially pure with signals of O-toluidine as an sp3-sp3 N-H band at 1634.00 cm-1 was present on the FTIR. For Benzoic acid and O-toluidine fractions, the purity level was very high as each specific FTIR band correlated to the structure of the compound. Benzoic acid has a specific sp2-sp2 C=O stretch band at 1965.60 cm -1 and 1915.07 cm-1 present, and O-toluidine had three sp3-sp3 N-H stretches present at 1618.21 cm-1, 3462.53 cm-1, and 3358.31cm-1.

Acid-Base Extraction resulted in the isolation of each three components, however with low % recovery; % recovery for O-toluidine was 0.57%, for Benzoic acid 1.17%, and for Anisole .95%. On the other hand, purification of each component after analyze of each FTIR Spectrums concludes that each component is highly pure as each FTIR Spectrum has the specific bands correlating to each specific compound and the melting point range of the recovered Benzoic Acid was concluded to be 123.3-126.0 ?°C which is approximately 2 degrees from the literature value of 122.0 ?°C.

Conclusion

Overall Acid-Base extraction as a means of separating and purifying organic compounds was successful at separating each three components with purity as each specific FTIR band correlated to the structure of the compound. Benzoic acid has a specific sp2-sp2 C=O stretch band at 1965.60 cm -1 and 1915.07 cm-1 present, and O-toluidine had three sp3-sp3 N-H stretches present at 1618.21 cm-1, 3462.53 cm-1, and 3358.31cm-1.

Also, Acid-Base extraction was not an efficient system to obtain a high % recovery. Overall % recovery was significantly low for all three components. Benzoic acid had the highest % recovery (1.17%) and O-toluidine had the lowest % recovery (0.57%).

Future goals to test the % recoveries of Acid-Base extraction could involve using a mixture of 1:1:1 ratio of O-toluidine, Benzoic Acid, and Anisole and using a 1:1 ratio of stronger acids and bases when converting the Bronsted- Lowry Acid and Base to their conjugate bases and acids.

Experimental

All reactants were reagent grade and provided by the University of Colorado Denver Chemistry Department. FTIR spectra were obtained with a Nicolet iS5 ATR FTIR spectrometer, and all measurements were in cm-1.

Isolation of o-toluidine:

Methylene chloride (10 mL) was added to a stock solution (10 mL, Anisole, Benzoic acid, O-toluidine). The organic solution was extracted with 3 M hydrochloric acid (3 x 20ml). The aqueous layers were combined and adjusted to 12 pH with 10 M sodium hydroxide (10 mL). The neutralized aqueous solution was extracted with dichloromethane (3x). The organic layer was dried over magnesium sulfate then collected via gravity filtration. The resultant organic solution was condensed in a tared scintillation vial in a hot water bath yielding reddish/yellowish oil, 0.856g (8.56 mmol, 0.57 %); FTIR (cm-1): 2931.97, 2862.49, 1498.20, 1311.42. Note thin layer chromatography plates developed in 1:1 Ethyl Acetate/ Hexane system.

Isolation of Benzoic acid:

5% sodium hydroxide 15 mL was added to the remaining stock solution. The organic solution was 22 ?°C with 6 M hydrochloric acid, five mL. The aqueous solution was allowed to cool in an ice bath until no more precipitation formed. The aqueous layer was collected via vacuum filtration. The resultant aqueous solution was collected in a tared a scintillation vial yielding white precipitate, 0.106g (1.06 mmol, 1.17 %); 123.30-126.0 ?°C (122 ?°C); 3067.77, 2935.13, 2862.49, 1965.60, 1915.07, 1675.06, 1602.42, 1586.63.

Isolation of anisole:

Methylene chloride (5 mL) was added to the organic layer from the previous extraction. The organic layer was dried over magnesium sulfate and then collected via gravity filtration. The organic layer was dried over magnesium sulfate then collected via gravity filtration. The resultant organic solution was placed in a tared scintillation vial in a hot water bath yielding reddish pinkish oil, .901 g (9.01 mmol, 0.95%). FTIR (cm-1): 3462.53, 3358.31, 1618.21, 3020.40, 2928.81, 2862.49, 1583.47. Note thin layer chromatography plates developed in 1:1 Ethyl Acetate/ Hexane system.

References

1. Padias, Anne. Making the Connections a How to Guide for Organic Chemistry Lab Techniques. Hayden McNeil Publishing. 2007. P 119.

2. Interpretation of Experimental Data. Web. 19 Oct. 2018 https://chemistry.syr.edu/totah/che276/support/6a1.handouts/4.datainterp.pdf

3. Thin Layer Chromatography. Web. 19 Oct. 2018 https://academics.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/thin_layer_chrom.html

4. Clark, Jim. Thin Layer Chromatography. June 2016. Web. 19 Oct. 2018 https://www.chemguide.co.uk/analysis/chromatography/thinlayer.html

5. Experiment 2 - Thin Layer Chromatography. P 2. Web. 19 Oct. 2018 https://webpages.uidaho.edu/chem276/files/2%20-%20Thin%20Layer%20Chromatography.pdf

6. Mohrig, Jerry, Christina Noring Hammond, Paul F Schatz. Techniques in Organic Chemistry. W.H. Freeman & Company, United States, 2009. 1st ed., pp. 151-162.

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